Angiotensin{hd II {B position 8 analogs

ABSTRACT

The present invention relates to novel angiotensin II derivatives which are particularly useful in inhibiting the action of angiotensin I and II.

United States Patent [1 1 Regoli et al.

[4 1 Sept. 23, 1975 [54] ANGIOTENSIN II POSITION 8 ANALOGS [75] Inventors: Domenico C. Regoli, Magog; Won

Kil Park, Sherbrooke, both of Canada [73] Assignee: University of Sherbrooke, Quebec,

Canada [22] Filed: Dec. 27, 1971 21 App1.No.: 212,257

[52] US. Cl 260/112.5; 424/177 [51] Int. Cl. C07C 103/52; A61K 37/26 [58] Field of Search 260/1125 [56] References Cited UNITED STATES PATENTS 3,751,404 8/1973 Sipos et al. 260/1125 FOREIGN PATENTS OR APPLICATIONS 1,155,925 6/1969 United Kingdom 260/1125 OTHER PUBLICATIONS Primary Examiner-Elbert L. Roberts Assistant ExaminerReginald .I. Suyat Attorney, Agent, or FirmCushman, Darby & Cushman [5 7] ABSTRACT The present invention relates to novel angiotensin II derivatives which are particularly useful in inhibiting the action of angiotensin I and II.

3 Claims, 4 Drawing Figures US Patent Sept. 23,1975 Sheet 3 of3 3,907,762

, ANGIOTENSIN POSITION 8 A NA LOGS PRIOR ART Angiotensin, my are natural substances correspond- Recently, both antiotensins haye beenlclassified as, hormones (Catt. K. J.: LANCET 1 827, 1970). It has also been found that they areliberated in the blood through the action of an enzyme of renal origin renin) on a plasmatic protein known as flangio't'e'n' sinogen. Amongst the biological effects of angioten'sin there maybe mentioned the vasoconst'r'icting effect which is considered most important to maintain suitable levels of arterial pressure or to produce hypertension. In view of this, the angiotensins have consequently been considered as one of the pathogenic factors causing arterial hypertension. 1

. A further physiological effectof angiotensins is their ability to stimulate the liberation of aldosterone from the adrenal cortex. The hypersecretion of ald osterone can cause a retention of sodium ions and water with accumulation of fluids in the extracellular space'ythis type of retention being known as oedema.

More recently, it has beenfound that angiotensins can stimulate the, liberation of 'catecholamine fromthe adrenal glands in man and in animals (Feldberg et al: J. Physiol. 171 98, l96 t). Further,angiotensinsare suspected to play an undesirable role in the'central-nervous system and the sympathetic nervous system. A

Many substances have been tried as, antagonists for angiotensins without satisfactory riesultjs, for example, lidophlazine, guancidine and osajin. Though these substances reduce the activity of'ang iotensinjs-in tissues or animals, the antagonism is neither specific njor 'competitive. The antagonism is mainly due to the property of these substances to depress the biological response of the tissues rather than -specifically antagonise the response to angiotensins. 7

Accordingly, because of the pathogeneticu'ole of angiotensins in hypertensive and oedematicdiseases it has been found highly desirable to develop pharmacologi'cal antagonists of angiotensins for blockingthe undesirable action of angiotensins. 3

THE INVENTION l '2"'3 '4 s ,6 Asp Arg Val Tyr Ile 5 His Q Pro wherein R is Leu (leucine),

lle (isoleucine),

Val (valine),'or a-amino-n-But (wamino-n-butyric acid) and Asp is aspartyl, Arg i s arginyl, Val is valyl, Tyr is tyrosyl, lle is isoleucyl, His is histidyl and Pro is prolyl. The compounds of the present invention have been found to be potent antagonists of angiotensin while maintaining little direct pressor effect, and with the ex; ception of 5-Ile-8-lle Angiotensin they are almost devoid of myotropic activity.The compounds are up to, times more potent than the recently published 8- Ala-Angiotensin Turker et al, Europ. J. Pharmacol. 285., 1971 and Park et al," Brit. J. Pharmacol. 43 418, 1971. I

The compounds of the present invention are competitive inhibitors of Angiotensin, and while two of them, 5 Ile -8-Ile Angiotensih and 5-Ile-8Leu-Angiotensin have relatively prolonged action in vivo.

PREPARATION OF COMPOUNDS The compounds of the present invention are prepared by the method of Merrifield described in J. Am. Chem. Soc. 85 2149, I963. In order to build up the peptide chain, the various amino acids must first be blocked to ensure, the interlinkage. This is done by forming the butyloxycarbonyl (Boc) derivative of the amino acid at the C-terminal. This is done generally by reactingeach amino acid with t-butoxycarbonylazide as described by Schwyzer et al in Helv. Chim. Act 42 following protected amino acids were prepared: Bocleucin'e l-l o, Boc-proline, Boc-isoleucine, Boc-valine and Boc-nitro ar ginine. Other protected amino acids readily available from Mann Research Laboratory, New York, USA. are Bocimidazol-benzyl-histidine, Boc-O-benzyl-tyrosine and BocB-benzyl-aspartic acid.

In the first linkage step the amino acid desired in the 8-position is combined with chloromethylated copolystyrene 2% divinylbenzene (available from Mann Research Laboratory, New York, U.S .A.). The amino acid can be Bocleucine-H O, Boc-valine, Boc-aamino-n-butyric acid or Boc-isoleucine. This reaction is carried out in the presence of a solvent made up of triethylamine in absolute ethanol and ether to provide the starting Boe-leucine-polymen,

The Boc-leucine polymer is then coupled with the next Boc-amino ac id, namely Boc-proline, through a series of Eycles involving deprotection, neutralization 5b and coupling with ;N,N-dicyclohexylcarbodiimide.

Briefly, the Boo group is cleaned with 1N HCl in glacial acetic acid, and,the Ieucine-polymer obtained is neutralized with triethylamine. Then the .Boc-proline is added with N,N-dicyclohexylcarbodiimide to form the Bod-proline-leucine-polymer. This series of deprotection, neutralization and coupling is then repeated with each protected amino acid until the protected 8-leuangiotensirifi derivative is obtained.

The desired ,Aspartyl-arginyl-valyl-tyrosyl-isoleucylhistidyl-prolyl-leucine also referred to as (5-Ile-8-Leu 'angiotensin is' then obtained by bubbling hydrogen bromide in anhydrous trifluoroacetic acid through the protected octapeptide whereby the polymer is removed and t he remaining protective groups are removed by reduction with hydrogen using palladium black as catalyst. .Thepurified polypeptide is obtained by chromatography on a column of Sephadex G-25 using nbutanolaceticacid-water (BAW) as the solvent. The

: 2622, 1959. In accordance with this procedure the average yield for each angiotensin derivative prepared varies between 50 to 65% based on the mmoles of C- terminal amino acid esterified on the polymer.

There is thus obtained the L-aspartyl-L-arginyl-L- valyl-L-tyrosy1-L-iso1eucyl-L-histidyl-L-prolyl-L-aamino-n-butyric acid, the L-aspartyl-L-arginyl-L-valyl- L-tyrosyl-L-isoleucyl-L-histidyl-L-proline-L-valine, the L-aspartyl-L-arginyl-L-valyl-L-tyrosy1-L-iso1eucyl-L- histidyl-L-prolyl-L-isoleucyne and L-aspartyl-L- arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L- histidyl-L-prolyl-L-leucine.

EXAMPLE 1 Aspartyl-arginyl-valyl-tyrosyl-isoleucyl-histidyl-prolylleucine (-lle-8-Leu) angiotensin A. Preparation of Boc-Leucine-H o Leucine (2.62 g, 20.0 mmoles) and magnesium oxide (1.65 g, 41.0 mmoles) were ground together in a mortar to give a small particle size. The mixture was stirred in a round bottom flask for two hours at room temperature and suspended in 70 ml of 50% aqueous dioxane. t-Butoxycarbonylazide (42.0 mmoles) was added, and the mixture stirred at room temperature for 40 hours and added to 100 ml of cold water. The insoluble residue was removed by filtration and the filtrate extracted with ether (3X70 ml) and the organic layer discarded. The aqueous phase was bubbled with N, gas to remove ether and cooled in an ice-bath, brought to pH 3 to 4 with cold 50% citric acid. The acidified suspension was extracted with four times 100 ml portions of ethylacetate. The combined extracts were washed with saturated NaCl and after drying over anhydrous Na SO the ethylacetate was removed under vacuum. The residue was crystallized from ethanol by addition of water. Recrystallization from 50% ethanol yield 85% of the pure product.

The compound was dried to a white crystal, not bygroscopic and soluble in organic solvents. Insoluble in water. Boc-proline, lsoleucine, Valine (NO,) Arginine, are prepared as described and yield 75-90% of the desired Boc-amino acid.

B. Boc-Leucine-Polymer A solution of 0.754 g (3 mmoles) Boc-leucine-H,O and 0.42 ml (3 mmoles) of triethylamine in 20 ml of absolute ethanol and ethylacetate (1:1) mixture was added to 3.0 g of the chloromethylated copolystyrene 2% divinylbenzene. The mixtue was stirred with a magnetic stirrer under reflux at 8090C for 24 hours. The esterified polymer was collected by filtration, washed with absolute ethanol-ethylacetate (1:1) mixture (100 ml), absolute ethanol (100 ml), water 100 ml) and absolute methanol 120 ml) and then dried under vacuum over P 0, and paraffin.

The amino acid analysis showed this substituted polymer to contain 0.52 mmoles of Boc-leucine/g of polymer.

C. t-Butyloxycarbonyl-B-benzyl-aspartyl-nitro-arginylvalyl-O-benzyl-tyrosyl-isoleucyl-imidazol-benzylhistidyl-prolyl-leucine polymer The Boc-leucine polymer (3.0 g, 1.56 mmoles) was placed in the reaction vessel (Park and Regoli, 1971) and the following cycle of deprotection, neutralization, and coupling was used to introduce each new residue: (1) washed with glacial acetic acid (3 X 50 ml); (2) 80 group was cleaved by 1.1N He] in glacial acetic acid (40 ml) for 40 min.; (3) washed with glacial acetic acid (3 X 50 ml); (4) washed with absolute ethanol (3 X 50 m1); (5) washed withdimethylformamide (DMF) (3 X 50 ml); (6) the hydrochloride neutralized with 8 ml of triethylamine in 45 ml of DMF for 10 min.; (7) washed with DMF (3 X 50 ml); (8) washed with methylene chloride (3 X 50 m1); (9) introduced 4 mmoles of the appropriate Boc-amino acid in 45 ml of methylene chloride and allowed to mix for 10 min.; (10) introduced 4.2 mmoles of N,N'-dicyclohexylcarbodiimide (Aldrich Chemical Co., Wis., USA.) in 5 ml of methylene chloride and the reaction mixture was shaken for 3 hours at room temperature and let stand in cold room overnight; (11) washed with methylene chloride (3 X 50 ml); 12) washed with absolute ethanol (3 X 50 ml). For Boc-nitro-arginine, and Boc-imidazolbenzylhistidine, step 8 was deleted and DMF was substituted for methylene chloride in steps 9-1 1. The protected octapeptide with polymer was dried in a desiccator over KOH, P 0 and paraffin in vacuo, dried weight: 4.7 g. D. Aspartyl-arginyl-valyl-tyrosyl-isoleucyl-histidylprolylleucine (5-lle, 8-Leu)-angiotensin The protected octapeptide (3.0 g) was suspended in 50 ml of anhydrous trifluoroacetic acid and HBr gas bubbled slowly through the suspension with occasional shaking for 50 min. at room temperature and under anhydrous condition. The reaction mixture was filtered and the polymer was washed (3 X 10 ml) with anhydrous trifluoroacetic acid. The volume of the combined filtrate was reduced in vacuo at 20. The peptide was precipitated by addition of anhydrous ether.

It was removed by filtration and washed with anhydrous ether several times. The partially protected octapeptide (1.59 g, 1.2 mmoles) was dissolved in 60 ml of mixed solvent methanol-acetic acid-water (10:3:1) and hydrogen was bubbled through the solution at atmospheric pressure for 38 hours using palladium black (1.9 g) as catalyst. The catalyst was removed by filtration and washed with the same solvent mixture (20 ml). The combined filtrates were evaporated to dryness in vacuo at 20. The residue was purified by chromatography on a column (4.5 X cm) of Sephadex G-25 coarse, using n-butanol-acetic acid-water (4: 1:5) as the solvent. Fraction of 10 ml each were collected, and from fraction 118-191 inclusive, 890 mg of peptide were obtained which was washed several times with anhydrous ether and dried over P 0 paraffin and KOH in vacuo. The yield is based on 1.56 mmoles of Bocleucine which. was esterified on the polymer.

Physical properties: white powder, soluble in water (2 mg/ml), insoluble in organic solvents. Analysis of the compound gave the following results:

mp 242245, PC; Rf (BAW) 0.48, Rf (BAPW) 0.45, TLC; Rf (BAW) 0.40, Rf (BAPW) 0.63, E;

1.13. Amino acid ratios on an acid hydrolysate; Asp, 1.02; Arg, 0.98; Val, 1.00; Tyr, 0.95; lle, 1.00; His, Pro, Len, 1.03. Anal. C I-1 N 0 2 20 (1048.19); C, 53.85; H,7.41; N,17.37. Found: C,

EXAMPLE 2 L-Aspartyl-L-arginyl-L-valyl-L-tyrosy1-L-isoleucyl-L- histidyl-L-prolyl-L-a-amino-n-butyric acid 5-Ile; 8-L-a-amino-n-butyric acid)-angiotensin,,

By following the procedure of Example 1 and starting with Boc-L-a-amino-n-butyric acid instead of Bocleucine-H O the above product is obtained. Analysis of the compound gave the following results:

mp 245 247, PC; Rf (BAW) 0.39, (BAPW) 0.33;

TLC: Rf (BAW) 0.26 Rf (BAPW) 0.57, E; 1.20. lo

Amino acid ratios on an acid hydrolysate; Asp, 1.05; Arg, 1.07; Val, 1.00; Tyr, 0.97; Ile, 1.00; His, 1.03; Pro, 0.98; a-amino-n-butyric acid, 1.02; Anal. C H N O .2 H O (1020.11): C, 52.98; H, 7.21; N, 17.85; Found: C, 53.02; H, 7.35; N, 17.96.

EXAMPLE 3 L-aspartyI-L-arginyI-L-valyl-L-tyrosyl-L-isoleucyl-L- histidyl-L-prolyl L-valine 5-Ile-5-val)-angiotensin,,

EXAMPLE 4 L-aspartyl-L-arginyl-L-valyl-L-tyrosyl-L-isoleucyl-L- histidyl-L-prolyl-L-isoleucine 5-Ile, 8Ile)-angiotensin By following the procedure of Example 1 and starting with Boc-isoleucine instead of Boc-leucine-H O the above product is obtained. Analysis of the compound gave the following results:

mp 230-234, PC; Rf (BAW) 0.46, Rf (BAPW) 0.47, TLC; Rf (BAW) 0.36, Rf (BAPW) 0.68, E; 1.14. Amino acid ratios on an acid hydrolysate; Asp, 0.98; Arg, 0.96; Val, 1.00; Tyr, 0.92; Ile, 2.04; His, 0.95; Pro, 1.00.

PHARMACOLOGY The products of the present invention have been tested for their potency on blood pressure, their myotropic effect on isolated organs, their antagonistic value in vivo and in vitro. Tests were made against angiotensin The antagonistic potencies of the compounds of the present invention were compared with that of 8- alanine angiotensin the latter compound having been described in Turker et al, Europ. J. Pharrnacol. 285, 1971 and Park et a1, Brit. J. Pharmacol. 43: 418, 1971.

The results of these tests are found in the graphs, wherein:

FIG. 1 shows the pressor effects of angiotensin (AT,,) and analogues on the blood pressure of ne phrectomized rats, anesthetized with urethane. The points are means i S.E. (vertical bars) of 6 experimerits. Abscissa: Log of the i.v. injected dose in g/kg Ordinate: Increase of blood pressure in mm Hg.

FIG. 2 shows the log dose response curves of AT on the rat blood pressure, in absence (continuous line) and in presence of 8-substituted analogues. Points rep.- resent the means of 6 experiments and vertical bars the SE. Abscissa: Dose of injected angiotensin in pg/kg. Ordinate: Increase of blood pressure in mm Hg.

FIG. 3 shows the myotropic effect of angiotensin (AT,,) in the absence (continuous line) and in the presence of increasing concentrations of antagonists. Points indicate means and vertical bars SE. of at least 3 experiments. (FIG. 3 is found on the same sheet as FIG. 1 Abscissa: Molar concentration of AT Ordinate: of maximal response. 1

FIG. 4 shows the log-log plots of dose ratios -1 (log(x-1)) against molar concentration of antagonist [B]. For each antagonist, the two points were calculated from the middle point (50% of maximum effect) of the curves reported in FIG. 3.

METHODS Direct effects and antagonistic potencies were tested in vivo (on the rat blood pressure) and in vitro (on the rat isolated stomach strip).

Rat Blood Pressure Male albino rats (250300 g), nephrectomized 24 hours before were anesthetized with urethane (1.4 g/kg s.c.). Blood pressure was recorded directly from the carotid artery with a mercury manometer and injections of the standard (5-Ile-angiotensin II) or of the new compounds were given into the jugular vein to measure the direct pressor efiect. For testing of antagonism, the standard doses of AT were given before, duringand after the intravenous infusion of one of the analogues. The infusions were administered through the femoral vein at a constantrate of 0.05 ml/min.

Rat Isolated Stomach Strip All experiments were preformed on rat stomach strips, suspended in a cascade system and superfused with 10 ml/min. of oxygenated (%-O and 5% CO Krebs solution at 37C. The contractions were recorded with an isotonic transducer and registered on a Harvard apparatus. A tension of 1.5 2 g was applied to the tissues. Maximal responses were obtained at the beginning of every experiment with a large dose (5-10 times higher than the ED-50) of standard S-Ileangiotensin II. Suitable doses of the standard were then given on order to obtain a curve dose response: thereafter, the antagonist was added to the perfusing fluid and the curve dose response of S-IIe-angiotensin II was repeated: if some antagonism was present, the doses of angiotensin II were increased to obtain maximum effect in the presence of antagonist.

The compounds used in the experiments are shown in table I.

TABLE I 1 2 3 4 5 6 7 8 Asp Arg V111 Tyr I11: His Pro Phe AT, (5-1lc-AT I Asp Arg V211 Tyr llu His Pro A111 1 (5-I1c- -Alu AT 11 Asp Arg Val T \-r Ilc His Pro n-But (5-11c-8-a-umino-n-But-AT 1II Asp Arg Val Tyr 11c His Pro Val (5-Ilc-8-Val-AT 1V Asp Arg Val Tyr Ile His Pro lle (5-1le-8-I1e-AT V Asp Arg Val Tyr Pm Leu 110 His The results are expressed as mean S.E. Statisticalsignificance was calculated with a students t-test for paired data.

RESULTS Rat Blood Pressure In a first series of experiments, increasing amounts of the analogues were injected intravenously to compare their pressor effects with those of standard AT Results are summarized in FIG. 1.

8-Ala-AT is almost inactive even when give in amounts 10,000 times higher than the minimum dose of AT The curve dose responses for the other 4 compounds lie between that of AT and that of 8-A1a-AT and do not show any significant difference between TABLE II Antagonism of the pressor efi'ect of AT by various 8 substituted analogues of AT Means and SE. of 6 experiments in bilaterally nephrectomized rats.

Agonist AFI'ER Antagonist. inj. Before During 90 ug/kg 0.2 16.5 i 1.0 13.2 :t l 2 8-A1a-AT 0.8 25.0 i 2.1 9.0 i 0.9 21.0 t 2 0 (2 #g/kg/min.) 2.0 12.0 i- 1.3 4. 15.0 i 1.1 0.2 10.7 i 0.9 12.4 i 1.4 8-a-amino-n- 0.8 19.5 i 1.1 5.0 i 0.3 20.2 i 1.8

But-AT 2.0 8.0 i 0.7 (2 pg/kg/min.) 4.0 12.3 i 0.7 8.0 15.3 i 0.8 0.2 16.2 1 0.9 9.0 i 0.5

8-val-AT,, 0.8 30.5 i 1.5 9.0 i 0.8 1" 5 i- 1.0 (1 /kg/min.) 2.0 11.0 i 0.7 I 4.0 15.8 i 1.2 8.0 22.0 i 1.6 0.2 12.4-31.2 5 8 10.9 10610.8

8-lle-AT 0.8 22.2 I 1.2 7.4 0.9 14.5 i 0 3 20.8 i 1.2

(0.6 #g/kg/min.) 2.0 11.4 i 1.3 4.0 15.4 t 1.7 8.0 21.2 I 2.4 0.2 10.2 0.9 5.5 i 0.7 9.3 i 0.9 8-Leu-AT 0.8 17.8 t 1.6 8.5 $0.5 18.8 i 1.8

(0.6 g/kg/min.) 2.0 8.5 1.3

Significance of differences between pressor effects of AT observed before and during infusion of the antagonist are given by P 0.01 11nd P 0.001

each other. Moreover, the log dose response curves are notparallel to that of AT The pressor effect of 8-aamino-n-But-AT and 8-Val-AT,, is quickly reversible like that of AT on the contrary, the increase of blood pressure eliced by 8-lle-AT and 8-Leu-AT lasts for about 7-15 min., especially when high doses (around 10 g/kg) are administered. I

In a second series of experiments, the analogues were tested for antagonism to AT the results are shown in FIG. 2.

Log dose response curves of AT are shifted to the right when the agonist is administered in the presence of the 5 compounds (I to V), shown in table 1.

Antagonistic effects of 8-Ala and 8-a-amino-n-But- AT are rapidly reversible (in less than 30 min.) while 8-Val-AT and even more 8-Ile and 8-Leu-AT depress the response to angiotensin for 60 or min. (table II). This indicates that lengthening of the aliphatic chain in position 8 may allow the compound to form a slowly reversible binding with the receptors. These interpretations are supported by the duration of the pressor effect with high doses of 8-I1e-AT and 8- Leu-AT (mean duration of pressor effect (20 mm Hg) in minutes corresponds to 2.7 i 0.2 min. for AT 4.1 i 0.4 min. for 8-Ile-AT and 14.6 i 4.4 min. for S-Leu- AT These results are means i SE. of 6 experiments.

Rat Isolated Stomach Strip For a more precised evalation of the interactions between angiotensin II and the new compounds at the receptors level, isolated rat stomach strips were used. As reported before (Regoli et al, Brit. J. Pharmacol. 23: 351, 1964), rat stomach strip is relatively sensitive to angiotensin II. In a cascade superfusion system, using oxygenated Krebs solution, the tissue does not show any important spontaneous activity: the responses to short infusions (3 min.) of increasing doses of AT consist of rapid contractions which remain constant at the maximum level obtainable with the dose. The responses are rapidly reversible after stopping the infusion. Sensitivity to angiotensin remains constant for several hours.

The compounds reported in table I were tested on this preparation for the direct myotropic effect and for antagonism against angiotensin II and S-hydroxytryptamine. The direct effect was evaluated as a percent of maximal response'obtainable with very high doses of each compound. As' shown in Table III, 8-Ile-AT maintains 30% of the intrinsic activity of AT while 8-Leu-AT has only and the other 3 compounds are almost inactive.

TABLE in Drug parameters of AT and analogues tested on rat isolated stomach strip).

a intrinsic activity and pD, (apparent affinity) according to Arlens, I964 "pK (30g K has been calculated by the equation 8 according to Furchgott, I967 To test the antagonism, the compounds II-V of Table I were added to the Krebs solution in concentration of 10 and 25 X 10' M. Ten minutes after beginning of the infusion, angiotensin was given again and the dose was increased to obtain full contraction. The results are summarized in FIG. 3.

8-Ala-AT 8-a-amino-n-ButAT and 8-Val-AT,, displace the log dose response curve of the agonist to the right: the curves remain parallel and do not show any important depression of the maximal response. Compounds II and III are about 10 times as active as 8-AIa-AT,, (FIG. 4). Compounds IV and V have an even stronger antagonistic potency and change significantly the slope of the log dose response curves. Moreover, after high concentrations of 8-Ile-AT and 8-Leu- AT the maximal response is slightly depressed (maximal residual response 87% after 8-Ile-AT and 89% after 8-Leu-AT p (see Table III) calculated from the log dose response curves, obtained in the presence of low concentrations of antagonists, is significantly higher for 8-Leu-AT and is increased for 8-Ile-AT The ratio agonist/antagonist and the percent of inhibition of AT for the 5 compounds are presented in Table IV.

TABLE IV Inhibition of the myotropic activity of AT and 5-I-IT on isolated rat stomach strip, by various 8 substituted analogues of AT ANTAGONIST AGONIST RATIO INHIBITION AG/ANT. (mean t S.E.)

S-Ala-AT AT 0.] 34 i 4 (6) I 17 i 6 (3) S-a-amino-n-But-AT AT I 39 i 7 (7) 10 10 i 4 (3) S-HT 0.02 0 (3) 8-Val-AT AT I 36 i 3 (6) l0 I5 2 4 (3) S-HT 0.04 0 (3) 8-Ile-AT AT 1 44 i 4 (3) I0 28 i 4 (3) 5-HT 0.04 4 i l (3) B-Leu-AT AT I 55 i 3 (3) IO 47 i 4 (3) S-HT 0.04 0 (3) In parenthesis, the number of individual determination.

This table reports also the absence of inhibition of S-hydroxytryptamine. The fact that 50% of inhibition of AT is obtained with 8-Leu-AT and 44% for 8-Ile- AT at the ratio agonist/antagonist of 1, indicates that two molecules of antagonist (8-Leu-AT and 8-Ile- AT are enough to prevent the action of one molecule of AT The absence of parallelism and the depression of the maximum effect after high doses of IV and V are suggestive for a slow dissociation from the receptors. This is confirmed by the high value of p for 8-Leu and 8-Ile-AT (see Table III).

TOXICITY STUDIES short periods of time (60-120 min.) because of the short half-life of the compounds. This type of application requires the control of the acute toxicity of the compounds in animals.

In view of the application in humans, several toxicity studies were performed.

The four compounds were administered intravenously (i.v.) at high doses to three animal species: rabbits (up to 5 mg/kg of each compound), rats (up to 20 mg/kg) and mice (up to 20 mg/kg).

The animals were watched during 6 hours following the i.v. administration to detect symptoms and signs of acute toxicity. The survival and the increase of the body weight were measured during 15 days following the i.v. administration, and compared with a control group (n= 10).

The results of one of these experiments using 5-Ile-8-Leu-angiotensin II are summarized in Table V.

TABLE V Arg is arginyl Acute toxicity of 5-lle-8-Leu-AT val "3 I Dose of Acutc Survival Species n compound symptoms after l5 Body wcight* Skin test ED-SO days Rabbit l mg/kg none 10091 2.5 g N.S. Negative 0.5 #g/kg i.\. Rut IO rng/kg I007: l.() g N.S. 0.2 #g/kg l.\'. Mice I0 20 rng/kg l0()'/( O.5 g N.Sv 0.2 #g/kg Mcnn increase with respect to control groups) N.S. Statistically not significantv The other three compounds (5-lle-8-a-amino-n-Butangiotensin ll, 5-lle-8-Val-angiotensin II and 5-lle-8 -lle-angiotensin ll) have similar effects to those shown for 5-lle-8-Leu-angiotensin II.

We claim:

1 As new derivatives of Angiotensin the L epimers corresponding to the general formula:

Asp Arg Val Tyr lle His wherein i Asp is aspartyl Pro- R 2 The compound of claim 1- wherein R is valine.

3. The compound of claim 1 wherein R is a-amino butyric acid. 

1. AS MEW DERIVATIVES OF ANGIOTENSIONII THE L EPIMERS CORRESPONDING TO THE GENERAL FORMULA:
 2. The compound of claim 1 wherein R is valine.
 3. The compound of claim 1 wherein R is Alpha -amino butyric acid. 